The invention relates to the use of a peroxiredoxin (thiol-specific antioxidant) and/or a xcex2-tubulin as a protective antigen against helminth parasites.
Each species of domestic animal can be parasitised by a number of different species of helminth, a process which usually causes disease. For example, the parasitic trematode Fasciola hepatica is known to be one cause of the economically important disease fascioliasis in ruminants, such as cattle and sheep. The parasite enters the mammalian host by penetrating the gut wall and spends approximately seven weeks feeding on and burrowing through the liver mass before migrating into the bile duct. Following infection, development of immunity in the host can be poor and resistance to reinfection in already infected hosts may be only partial or non-existent. Other parasitic flukes include Fasciola gigantica and Dicrocoelium spp., Paramphistomum spp. and also Schistosoma spp., eg S. bovis and S. mansoni. 
Problems are also caused by nematodes such as hookworms (e.g. Necator, Ancylostoma, Uncinaria and Bunostomum spp.).
Of the blood feeding nematodes the genus Haemonchus causes anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated.
Other parasitic worms of economic importance include the various species of the following helminth genera:xe2x80x94
Trichostrongylus, Nematodirus, Dictvocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris and Strongylus. In addition to domestic livestock, companion animals and humans may also be infected, not infrequently with fatal results and helminth infections and infestations thus pose a problem of considerable worldwide significance.
Control of helminth parasites of grazing livestock currently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques are often unsatisfactory, firstly because anthelmintic drugs may have to be administered frequently, secondly because resistance against anthelmintic drugs is becoming increasingly widespread and thirdly because appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
Numerous attempts have been made to control helminth parasites of domestic animals by immunological means. With very few exceptions (e.g. the cattle lungworm, Dictyocaulus viviparus) this has not proved possible.
A vaccine against F. hepatica has been proposed in WO90/08819 comprising a glutathione-S-transferase from F. hepatica as antigenic material. Further vaccines against F. hepatica have been proposed in WO94/09142, WO94/28925 and PCT/GB95/02350 comprising respectively a Cathepsin L, a dipeptidyl peptidase and a class of haemoproteins from F. hepatica as antigenic material.
Bennett (UK Patent No. 2169606B) extracted various antigens from Fasciola organisms by a process which separates antigens specific to the juvenile stage from antigens present throughout the juvenile and adult stages.
Furthermore crude in vitro excretory/secretory (E/S) products can under some circumstances confer immunity on rats (Rajasekariah et al, Parasitol. 79 (1979), p. 393-400).
It has now been found that animals vaccinated against F. hepatica using a relatively impure haemoprotein preparation, the pure counterpart of which is described in PCT/GB95/02350, produce antibodies against peroxiredoxin and xcex2-tubulin molecules of fluke origin. This discovery opens up the possibility of vaccines against F. hepatica and other helminths based on the use of peroxiredoxin and/or xcex2-tubulin molecules and/or corresponding proteins produced by other helminth parasites as antigens.
Accordingly an aspect of the present invention provides a vaccine composition for use in combating a parasitic infestation of helminths in a mammal wherein the antigenic material comprises a peroxiredoxin and/or a xcex2-tubulin molecule, in at least partially purified form, or an antigenic fragment or epitope, component, precursor, analogue, variant or functionally equivalent derivative thereof, together with a carrier and/or adjuvant.
The invention also provides a method of combating a parasitic infestation of helminths in a mammal comprising administering to said mammal a vaccine according to the invention as hereinbefore defined in an amount effective to combat said infestation.
Alternatively viewed, the invention provides for the use of the molecules as hereinbefore described in the preparation of a vaccine composition for combatting a parasitic infestation of helminths in a mammal.
The mammal is preferably a ruminant, for example cattle or sheep, but the vaccine and method of the invention may also find application in humans, companion animals such as dogs and cats or in other domestic animals.
Preferably the peroxiredoxin and/or xcex2-tubulin molecules are derived from flukes such as Fasciola or Dicrocoelium, in particular from the liver fluke Fasciola hepatica. Alternatively it is preferred that the peroxiredoxin and/or xcex2-tubulin molecules should be capable of stimulating an immune response which will be effective against Fasciola or Dicrocoelium, in particular F. hepatica and F. gigantica, such peroxiredoxin and/or xcex2-tubulin molecules from other species as are capable of conferring a cross-protective immune response thus forming a particularly preferred aspect of the invention.
The F. hepatica peroxiredoxin and xcex2-tubulin molecules shown hereinafter to possess cDNA sequences and predicted amino acid sequences as shown in FIGS. 2 and 4 respectively are particularly preferred for use in the vaccine and method of the invention.
The peroxiredoxin and/or xcex2-tubulin molecules incorporated in the vaccine according to the invention are in at least partially purified form. Preferably the molecules of the present invention are at least 75% pure and more preferably at least 95% pure. It will be appreciated that once peroxiredoxin and/or xcex2-tubulin molecules of at least 95% purity have been obtained they can be admixed with one or more further purified antigenic proteins, to form a polyvalent vaccine.
According to the present invention the peroxiredoxin and/or xcex2-tubulin molecules incorporated in the vaccine may be in the form of antigens, antigenic fragments, epitopes, components, precursors, analogues or functionally-equivalent derivatives thereof.
A preferred form of polyvalent vaccine according to the invention will contain peroxiredoxin and/or xcex2-tubulin polypeptides as referred to above in combination with a Cathepsin L-type antigen as described in more detail in International Patent Application No. WO94/09142 or a dipeptidyl peptidase antigen as described in more detail in International Patent Application No. WO94/28925 or a class of haemoprotein molecules as described in more detail in International Patent Application No. PCT/GB95/02350. Thus the Cathepsin L and/or dipeptidyl peptidase and/or haemoproteins are preferably derived from flukes such as Fasciola or Dicrocoelium, in particular the liver fluke F. hepatica. Such a polyvalent vaccine will, by inducing immunity in the host species against two or more separate aspects of the invading helminth parasite, significantly increase the likelihood of protection against the helminth and significantly reduce the chances of infestation occurring.
Monovalent vaccines according to the invention may also have an anti-fecundity effect on helminth parasites, and this effect should be still more marked with polyvalent vaccines.
In a preferred aspect the polyvalent vaccine comprises peroxiredoxin and/or xcex2-tubulin polypeptides according to the present invention together with a Cathepsin L1 having molecular weight of 27 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis as disclosed in WO94/09142 and/or a Cathepsin L2 having molecular weight of 29.5 kDa by the same technique as disclosed in WO94/09142 and/or a dipeptidyl peptidase having molecular weight of 200 kDa by the same technique as disclosed in WO94/28925 or one or more of a class of haemoproteins of at least 200 kDa by gel filtration chromatography as disclosed in PCT/GB95/02350.
The vaccines according to the invention may be formulated with conventional carriers and/or adjuvants and the invention also provides a process for the preparation of the vaccines comprising bringing into association purified peroxiredoxin and/or xcex2-tubulin molecules as hereinbefore described and one or more adjuvants or carriers. Suitable adjuvants include aluminium hydroxide, saponin (ISCOMs), quil A and more purified forms thereof, muramyl dipeptide, mineral and vegetable oils, DEAE dextran, nonionic block copolymers or liposomes such as Novasomes (Trade Mark of Micro Vesicular Systems Inc.), in the presence of one or more pharmaceutically acceptable carriers or diluents. Carriers for peptide sequences corresponding to epitopes of peroxiredoxin or xcex2-tubulin molecules according to the invention can be proteins such as Hepatitis B core antigen multiple antigen peptide or lipopeptides such as tripalmitoyl-S-glycerylcysteinylserylserine (P3CSS). Suitable diluents include liquid media such as saline solution appropriate for use as vehicles. Additional components such as preservatives may be included.
Administration of the vaccine to the host species may be achieved by any of the conventional routes, e.g. orally or parenterally such as by intramuscular injection, optionally at intervals e.g. two injections at a 7-35 day interval. A suitable dose when administered by injection might be such as to give an amount of protein within the range 10-500 xcexcg.
According to a further aspect, the invention provides the F. hepatica peroxiredoxin molecule or antigenic fragments, epitopes, components, precursors, analogues or variants thereof and functionally-equivalent derivatives thereof having protective antigenic activity against one or more helminth parasites, characterised by:
(a) having at least a portion which substantially corresponds to the amino acid sequence as shown in FIG. 4;
(b) being encoded by a nucleotide sequence at least a portion of which substantially corresponds to the sequence shown in FIG. 4;
While the peroxiredoxin and/or xcex2-tubulin molecules for use in the vaccine according to the invention may be prepared by isolation from the helminths, it may also be convenient to prepare them by recombinant DNA techniques with the known advantages which such techniques give in terms of scaling-up of production and reproducibility. Thus the invention also provides for peroxiredoxin and xcex2-tubulin molecules as hereinbefore described, produced by means of recombinant DNA techniques.
Accordingly, in one aspect, the present invention provides for nucleic acid sequences which encode the peroxiredoxin or the xcex2-tubulin molecules of the invention or antigenic portions thereof substantially corresponding to all or a portion of the nucleotide sequences as-shown in FIG. 4 for peroxiredoxin and FIG. 2 for xcex2-tubulin or sequences encoding helminth peroxiredoxin or xcex2-tubulin antigens which are substantially homologous or which hybridise with any of said sequences.
A nucleic acid according to the invention may thus be single or double stranded DNA, cDNA or RNA.
Variations in the peroxiredoxin or xcex2-tubulin-encoding nucleotide sequences may occur between different strains of helminth within a species, between different stages of a helminth life cycle (e.g. between larval and adult stages), between similar strains of different geographical origin, and also within the same helminth. Such variations are included within the scope of this invention.
xe2x80x9cSubstantially homologousxe2x80x9d as used herein includes those sequences having a sequence identity of approximately 50% or more, eg. 60% or more, and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By xe2x80x9cfunctionally equivalentxe2x80x9d is meant nucleic acid sequences which encode polypeptides having anti-oxidant or xcex2-tubulin functionality which are similarly immunoreactive i.e. which raise host protective antibodies against helminths.
Nucleic acid molecules which hybridise with the sequences shown in FIGS. 2 and 4 or any substantially homologous or functionally equivalent sequences as defined above are also included within the scope of the invention. xe2x80x9cHybridisationxe2x80x9d as used herein defines those sequences binding under non-stringent conditions (6xc3x97SSC/50% formamide at room temperature) and washed under conditions of low stringency (2xc3x97SSC, room temperature, more preferably 2xc3x97SCC, 42xc2x0 C.) or conditions of higher stringency eg. 2xc3x97SSC, 65xc2x0 C. (where SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).
Methods for producing such derivative related sequences, for example by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids are well known in the art, as are methods for determining whether the thus-modified nucleic acid has significant homology to the subject sequence, for example by hybridisation.
Provision of a nucleic acid molecule according to the invention thus enables recombinant peroxiredoxin or xcexc-tubulin or immunogenic fragments thereof, to be obtained in quantities heretofore unavailable, thereby permitting the development of anti-helminth vaccines.
In another aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences encoding one or more polypeptides capable of raising protective antibodies against helminth parasites, which sequences incorporate one or more antigenic determinant-encoding regions from the peroxiredoxin or xcex2-tubulin encoding sequences as shown in FIGS. 2 and 4.
The present invention also extends to synthetic polypeptides comprising one or more amino acid sequences constituting a peroxiredoxin or xcex2-tubulin molecule or antigenic portions thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in FIGS. 2 and 4 or a functionally-equivalent variant thereof.
Additional aspects of the invention related to the above include vectors containing one or more nucleotide sequences as defined above; host cells, for example bacteria such as E. coli or yeast cells such as Saccharomyces spp., or more preferably eukaryotic cells, transformed by such vectors, for example by a baculovirus vector; and processes for preparing recombinant peroxiredoxin and xcex2-tubulin polypeptides or antigenic fragments or epitopes thereof comprising culturing such transformed host cells and isolating said peroxiredoxin or xcex2-tubulin polypeptides or fragments or epitopes from the cultured cells.
An alternative live or inactivated vaccine formulation may comprise an attenuated or virulent virus or a host cell, e.g. a microorganism such as a bacterium, having inserted therein a nucleic acid molecule (e.g. a DNA molecule) according to the invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule. A bacterial vector which elicits local gut mucosal immunity to a fluke antigen which then blocks juvenile fluke migration is particularly preferred, notably invasive species such as Salmonella species.
Additional antigenic materials may also be present in the vaccine thus giving an enhanced protective effect against the helminth parasite in question or a combined protective effect against one or more additional parasitic infestations.
A yet further aspect of the invention provides monoclonal or polyclonal antibodies capable of inducing immunity to peroxiredoxin or xcex2-tubulin molecules in a mammal when administered to said mammal, the antibodies having an affinity for the variable region of one or more further antibodies, said further antibodies having an affinity for said thiol-specific antioxidant or xcex2-tubulin molecules.
This approach, the so-called xe2x80x9canti-idiotypexe2x80x9d approach, permits formulation of a vaccine which will dispense entirely with the original antigen and may offer even greater advantages in terms of safety, avoidance of side effects and convenience of manufacture.